The present invention lies in the field of protein chemistry.
The determination of the total protein present within a solution is important in many analytical procedures such as in the determination of the purity of a protein and in determining the protein chemistry of a blood or urine sample. Four spectroscopic methods are routinely used to make such determinations. These include measurement of the protein""s intrinsic ultraviolet (U.V.) absorbance, and three methods which generate a protein-dependent color change, namely, the Lowry assay, the Smith copper/bicinchoninic assay, and the Bradford dye assay, U.S. Pat. No. 4,023,933. Although one or more of these methods is used routinely in almost every biochemical laboratory, none of these procedures are particularly convenient for the following reasons.
The first, U.V. absorbance, has limited application because it requires a pure protein with know extinction coefficient in a solution free of interfering substances. The Lowry and copper/bicinchoninic assays require the preparation of several reagent solutions which must be carefully measured and mixed during the assay. This is followed by lengthy, precisely timed incubations at closely controlled elevated temperatures, then immediate absorbance measurements of the unstable solutions. Both assays may be affected by other substances frequently present in biochemical solutions, including detergents, lipids, buffers and reducing agents. To control for these factors each assay must also include a series of standards, each with a different, known concentration of added protein but otherwise having the same composition as the sample solutions. The Bradford dye assay is faster, involves fewer mixing steps, does not require heating, and gives a more stable calorimetric response than the two previous assays. Like them, however, its response is prone to influence from nonprotein sources and protein standard solutions must be used.
The preparation of protein standards is tedious and error-prone. Protein preparations, often of bovine serum albumin, are available as preweighed powder or in sterile solutions of measured concentration, but their use in preparing standard solutions still requires several precise measurements and dilutions. Frequently this involves compromises between convenience, precision, and the requirement that the standards contain the same concentrations of non-protein components as the sample solutions.
U.S. Pat. No. 5,496,737 (hereinafter the ""737 patent) discloses a process for producing a solventless protein assay standard, which largely solved the problems of the prior art. This patent teaches a method for preparing convenient and stable protein assays and protein assays with included standards. Even though the process of the ""737 patent solved many of the problems associated with previous protein assays, the process and the resultant preparations still had a number of problems which made the process and product not quite suitable for mass production and commercial use. The most serious of these is that the primary buffer component of the anhydrous dye reagent mixture used in the solventless protein assay is phosphoric acid. This causes the dye reagent mixture to have the properties of a viscous liquid upon drying. Because of this, if the plate is inverted during storage or shipping, the anhydrous reagent mixture may slowly flow out of the receptacle or onto the cap or tape seal. This could cause the dye reagent to be lost when the cap or tape was removed, and thereby decrease the colorimetric response of the assay. It also makes it difficult to prevent any added protein standards from mixing with the dye reagent. Although this is normally not a problem, as the anhydrous protein shows little or no reaction with the anhydrous dye reagent, in the presence of water the acidic dye reagent catalyzes protein hydrolysis. Because the dye does not bind to the small peptides or individual amino acids produced by protein hydrolysis, if water or water vapor were allowed to enter the receptacle before it was used in an assay, this could cause the colorimetric response to the protein to decrease when an assay was performed.
Thus, there is a need for a solventless dye reagent and a solventless protein assay preparation in which the buffering agent does not remain liquid, but is crystallized or forms a solid upon drying.
The present invention fills this need by providing for a dye assay reagent mixture comprised of a protein reacting dye and a solidifying agent such as a solid acid.
The present invention further provides for method for preparing a solventless dye assay reagent comprising preparing a dye solution containing a dye, a dye solvent and a solidifying agent; placing an aliquot of the dye solution within a receptacle; removing the dye solvent under conditions wherein the solidifying agent precipitates, crystallizes or solidifies. Preferred dyes are those listed in Table 1. A most preferred dye is Coomassie blue G250 (C.I. 42655). In a preferred embodiment, the solidifying agent is a solid acid. Preferred solid acids are those listed in Table 2. In even a more preferred method of the present invention a stabilizer is added such as those listed in Table 3.
The present invention further provides for a solventless protein assay standard comprised of a protein reacting dye and a solidifying agent such as a solid acid, and a solventless protein.
The present invention further provides for a method for preparing a solventless protein standard. The standard is prepared by preparing a dye solution containing a dye, a dye solvent and a solidifying agent, placing an aliquot of the dye solution within a receptacle, removing the dye solvent under conditions wherein the solidifying agent solidifies. A protein solution containing a known amount of protein and a known amount of protein solvent is prepared; a known amount of the protein solution is placed within the receptacle containing the dye such that the amount of protein within the receptacle can be calculated, and the protein solvent is removed. The preferred dyes are listed in Table 1; and the preferred dye is Coomassie blue G250 [Color Index (C.I. 42655]. Preferred solidifying agents are solid acids particularly, those listed in Table 2, particularly sodium bisulfate. In a preferred embodiment a stabilizer is added such as those in Table 3.
In an alternative process the order in which the dye reagent and protein are placed in the receptacle is reversed. In this embodiment, the solventless protein acid standard is prepared by preparing a protein solution containing a known amount of protein and a known amount of protein solvent. A known amount of the protein solution is placed within a receptacle such that the amount of protein within the receptacle can be calculated. The protein solvent is removed. A dye solution containing a dye, dye solvent and a dissolved solid acid is prepared. An aliquot of the dye solution is placed within the receptacle containing the protein the dye solvent removed under conditions wherein the solid acid precipitates, crystallizes or solidifies. The preferred dyes, solidifying agents and stabilizers are the same as listed above.
The present invention further provides for solventless, protein-assay standard comprised of a receptacle, said receptacle containing a solventless dye reagent with a solidifying agent and a dehydrated protein wherein upon addition of an appropriate solvent the dye reagent and protein react together to produce a detectable color indicating the presence and/or concentration of protein.
The present invention further provides for an apparatus comprised of a series of receptacles such as a multiwell or microtiter plate wherein one or more of the receptacles contain a known concentration of dehydrated protein, a protein-reactive reagent and a solidifying agent such that upon addition of an appropriate solvent, the reagent and protein react to indicate the presence and/or concentration of protein.
The present invention further provides for one or more of the receptacles contain only the dye and solidifying agent and one or more of the receptacles contain the dehydrated protein, the protein-reactive reagent and solidifying agent.